Cucumis melo extract coated and/or microencapsulated in a fat-soluble agent based on a fatty substance

ABSTRACT

The present invention relates to a composition comprising an active plant extract containing superoxide dismutase, said extract being coated and/or microencapsulated in a fat-soluble agent based on a fatty substance.  
     It also relates to its method of manufacture, to the pharmaceutical, cosmetic or food compositions containing it and as a medicament, in particular, in protecting the epidermis against UV radiation and allergies.

[0001] The present invention relates to a novel composition comprisingan active plant extract containing superoxide dismutase, in particular aprotein extract of cucumis melo. It relates in particular to activeplant extracts coated and/or microencapsulated in a fat-soluble agentbased on a fatty substance and which may be useful in oral or topicalapplication.

[0002] While oxygen is essential for us to function, it neverthelessgenerates toxic reactive oxygen species which have a negative effect onour body.

[0003] In biological systems, reactive oxygen species comprise freeradicals such as the superoxide radical (0₂.⁻), the hydroxyl radical(OH.), nitrogen monoxide (NO.) or peroxyl radicals of lipid origin(L-OO.). These free radicals are atoms or molecules whose electronconfiguration is characterized by the presence of an unpaired electron.This characteristic feature makes them unstable and, to becomestabilized, they can rapidly oxidize new biological molecules such asnucleic acids (DNA), enzymatic proteins or membrane lipids andparticularly polyunsaturated fatty acids (PUFA). The superoxide ion isthe free radical most commonly and most abundantly generated in cells.It therefore represents a considerable danger for the body. Its toxicitydepends in particular on its transformation to other more aggressivereagents such as the hydroxyl radical (OH.).

[0004] Our body acts constantly against the formation of free radicalsand several lines of defense may be envisaged for detoxifying the cell.

[0005] The first line of defense is provided by an enzyme calledsuperoxide dismutase (SOD). Three forms of SOD exist: one containingcopper and zinc, ie. Cu/Zn SOD; another containing manganese (Mn SOD)and finally one SOD containing iron (Fe SOD°). SOD plays a key role incombating free radicals since it allows the elimination of thesuperoxide ion. Superoxide dismutases are enzymes capable of inducingdismutation of the superoxide ions according to the reaction:

20₂+2H⁺→H₂O₂+O₂

[0006] This elimination of the superoxide ion leads nevertheless to theformation of hydrogen peroxide (H₂O₂) which is also toxic for the cell.The action of SOD should therefore be supplemented by a second line ofdefense which eliminates H₂O₂.

[0007] The second line of defense is provided by two enzymes: catalaseand selenium-dependent glutathione peroxidase (SeGPx) which destroysH₂O₂.

[0008] Under normal biological conditions, the human body constantlyproduces, but in low quantities, free radicals which are immediatelyneutralized by the existing defense systems. In some cases, if theproduction of free radicals increases (tobacco, stress, pollution, solarradiation and the like) and/or if there is a deficiency in antioxidantsubstances, this leads to an oxidant/antioxidant imbalance. Thisimbalance is increasingly closely correlated with numerous pathologicalconditions and with numerous imbalances. Because of this, the search forantioxidant substances has been a subject of constant interest in thesector of cosmetology, agro-foodstuffs or medicine.

[0009] Patent FR 2 287 899 has described, for example, the applicationin cosmetology of superoxide dismutase enzymes and in particular the useof these enzymes in the preparation of cosmetic compositions for skinand hair care.

[0010] SOD has therefore been used in pathological conditions induced byfree radicals, in particular in the case of chronic inflammations suchas, for example, in the treatment of Crohn's disease (Emerit et al.,1991, Free Rad. Res. Comms, 12-13, 563-569) or in the case ofradio-induced fibroses (Delanian et al., 1994, Radiotherapy andOncology, 32, 12-20).

[0011] These superoxide dismutases are in particular extracted frombovine erythrocytes (Markovitz, J. Biol. Chem., 234, p. 40, 1959), fromEscherichia Coli (Keele and Fridovitch, J. Biol., 245, p. 6176, 1970)and from marine bacterial strains (patents FR 2 225 443 and FR 2 240277).

[0012] Nevertheless, for reasons linked in particular to the infectioussubstances which these materials may contain, there is currently atendency to replace them with substances obtained from the plant kingdomwhich are reputed to be more healthy. These antioxidant enzymes areindeed present in variable quantities in some plants.

[0013] Thus, patent application FR 2 716 884 describes a protein extractof Cucumis melo having improved superoxide dismutase enzymatic activity,their method of preparation and their use in pharmaceutical or cosmeticcompositions for external topical use. However, such a protein extractis difficult to use as the enzymes which it contains (superoxidedismutase in particular) are inactivated in the presence of externalattacks (UV in particular) or while taking in formulations for oral use(gastric juice). It is therefore advisable to develop specificformulations which offer this protection so as to optimize the action ofthe antioxidant active agents.

[0014] To increase the plasma half-life of the free forms of SOD (Cu/Zn,Fe or Mn), various modified forms for parenteral administration havebeen proposed; there may be mentioned SODs conjugated with polyethyleneglycol (SOD-PEG), SODs conjugated with heparin (SOD-Heparin), SODsconjugated with albumin (SOD-albumin), polymers or copolymers of SOD andliposomal SODs. However, these various SODs have the major disadvantageof being only very slightly absorbed when they are administered by theoral route.

[0015] patent application FR 2 729 296 also describes a particularpharmaceutical composition which is well suited to oral administrationof superoxide dismutase essentially comprising, in combination, asuperoxide dismutase and a compound selected from the group consistingof ceramides, prolamines and polymeric films based on said prolamines.However, such a composition is not suitable for use by the topical routeand, in addition, cannot be used by people allergic to gluten.

[0016] The inventors have discovered, surprisingly, that when an activeplant extract containing superoxide dismutase (SOD) was coated and/ormicroencapsulated in a fat-soluble agent based on a fatty substance, itcould be used by the topical or oral route and its active agents, inparticular SOD, were protected against external attacks (UV or gastricjuice for example).

[0017] The present invention therefore relates to a compositioncomprising an active plant extract containing superoxide dismutase, saidextract being coated and/or microencapsulated in a fat-soluble agentbased on a fatty substance.

[0018] The expression “active plant extract containing superoxidedismutase” is understood to mean, for the purposes of the presentinvention, any extract obtained from plants having superoxide dismutaseenzymatic activity. Advantageously, it is a protein extract of cucumismelo, in particular in powdered form, advantageously freeze-dried. Moreadvantageously still, the cucumis melo is a progeny of the cell line95LS444 or of one of the hybrid lines derived from 95LS444.

[0019] In a particular embodiment, the superoxide dismutase enzymaticactivity of the plant extract is at least equal to 5 enzymatic units permg of extract, advantageously at least equal to 50 enzymatic units permg of extract.

[0020] The plant extract may be obtained by methods well known topersons skilled in the art, in particular by the method described inpatent application FR 2 716 884. It is also commercially available fromthe company BIONOV under the trademark EXTRAMEL®.

[0021] Advantageously, the plant extract according to the presentinvention may, in addition, have a catalase enzymatic activity. It mayalso comprise a coenzyme Q10, vitamins, lipoic acid, gluthathione,inorganic elements present in the plant, such as potassium, magnesium,calcium and selenium, and the like.

[0022] The expression “fat-soluble agent based on a fatty substance” isunderstood to mean, for the purposes of the present invention, any agentcontaining at least one fatty substance and having an index of balancebetween the hydrophilic part and the lipophilic part of the molecule(HLB) of less than 10.

[0023] Advantageously, the fat-soluble agent according to the presentinvention has a melting point of between about 40 and about 80° C., moreadvantageously still of between about 55 and about 60° C.

[0024] In a particular embodiment, the fat-soluble agent according tothe present invention is of plant origin. Advantageously, it is chosenfrom the group consisting of hydrogenated oils; palm oil or oil from theheart of palm fruit; hydrogenated seeds; stearates, in particular chosenfrom stearins, stearic acid and its derivatives; waxes; fatty acidmonodiglycerides; saturated C₁₄-C₂₀ fatty acid triglycerides andmixtures thereof.

[0025] Advantageously, the fat-soluble agent according to the presentinvention is hydrogenated oil based on plant oil, in particular chosenfrom the group consisting of hydrogenated coconut oil, hydrogenated palmoil, hydrogenated soybean oil or hydrogenated rapeseed oil and mixturesthereof. More advantageously still, it is hydrogenated palm oil.

[0026] It is also possible for the fat-soluble agent to containwater-soluble substances of the sugar or polyol type.

[0027] In a particular embodiment, the plant extract according to theinvention is present at a concentration of between 1 and 90% by weightrelative to the total weight of the composition, advantageously between20 and 50% by weight relative to the total weight of the composition.

[0028] The present invention also relates to a method for manufacturingthe compositions according to the present invention comprising thefollowing steps:

[0029] a) melting the fat-soluble agent,

[0030] b) depositing the liquid obtained in step a) on the active plantextract containing superoxide dismutase.

[0031] Advantageously, the plant extract is present of the form of apowder and step b) is carried out in a fluidized bed, moreadvantageously still at low temperature and a low moisture content.

[0032] In a particular embodiment, the temperature of step b) is lessthan the melting point of the fat-soluble agent and is advantageouslyabout 40° C.

[0033] The present invention also relates to a pharmaceutical orcosmetic composition comprising a composition according to the inventionand an excipient appropriate for oral or topical use for human or animaladministration. These compositions may be formulated for administrationto mammals, including humans. The dosage varies according to thetreatment and according to the condition in question.

[0034] In a particular embodiment, the pharmaceutical or cosmeticcomposition according to the present invention may be formulated foradministration by the oral route. The active ingredient may beadministered in unit forms for administration, mixed with conventionalpharmaceutical carriers, to animals or to human beings. The appropriateunit forms for administration comprise the forms such as tablets,gelatin capsules, powders, granules and oral solutions or suspensions.Advantageously, it comprises, in addition, yogurt and/or fibers, moreadvantageously still so as to form a homogeneous mixture with thecomposition comprising an active plant extract containing superoxidedismutase, said extract being coated and/or microencapsulated in afat-soluble agent based on a fatty substance according to the presentinvention.

[0035] In another particular embodiment of the invention, thepharmaceutical or cosmetic composition may also be formulated foradministration by the topical route. They may be provided in the formswhich are customarily known for this type of administration, that is tosay in particular lotions, foams, gels, dispersions, sprays, shampoos,serums, masks, body milks or creams for example, with excipientsallowing in particular skin penetration so as to improve the propertiesand the accessibility of the active ingredient. These compositionsgenerally contain, in addition to the composition according to thepresent invention, a physiologically acceptable medium, in general basedon water or solvent, for example alcohols, ethers or glycols. They mayalso contain surfactants, preservatives, stabilizing agents,emulsifiers, thickeners, other active ingredients leading to anadditional or potentially synergistic effect, trace elements, essentialoils, perfumes, colorants, collagen, chemical or mineral filters,moisturizing agents or thermal waters.

[0036] The present invention also relates to a composition according tothe present invention as a medicament.

[0037] Advantageously, it relates to a composition according to thepresent invention as a medicament intended for treating or forpreventing allergies or for protection the epidermis from UV radiation.

[0038] The present invention also relates to the use of a compositionaccording to the present invention for the manufacture of a medicamentfor topical use intended for treating allergies, eczema, vitiligo,psoriasis, lupus, skin fibrosis, for eliminating melanomas, forimproving cicatrizations of transplants, for preventing allergies or forprotecting the epidermis from UV radiation.

[0039] It also relates to the use of a composition according to thepresent invention for the manufacture of a medicament intended forprotecting the epidermis from UV radiation, for treating or preventingallergies, for treating asthma, anemia, male sterility, inflammatorydiseases such as anthropathy or degenerative diseases due to cellularand/or organic degeneration, in particular chosen from the groupconsisting of Crohn's disease, Parkinson's disease, Alzheimer's disease,colorectal cancers, fibroses of varied origins, degenerations due to aninfectious agent such as AIDS or hepatitis C or degenerations linked tothe use of a medicament.

[0040] The present invention also relates to the use of a foodcomposition comprising the composition according to the presentinvention as an energy source, as a tonic source or as an aid forrecuperation and for regaining fitness.

[0041] It relates, furthermore, to a composition according to thepresent invention as a medicament, in particular for topical use,intended for treating or for preventing allergies, advantageously forprotecting the epidermis against allergies.

[0042] It finally relates to the use of a composition according to thepresent invention for the manufacture of a medicament, in particular fortopical use, intended for treating or for preventing allergies,advantageously for protecting the epidermis against allergies.

[0043] Other objects and advantages of the invention will becomeapparent for persons skilled in the art through references to thefollowing illustrative drawings.

[0044]FIG. 1 represents a skeleton diagram of the process for thepreparation of the compositions according to the present invention.

[0045]FIG. 2 represents the effects evaluated 72 hours after irradiationof the composition of the present invention administered 24 hours beforeirradiation on keratinocytic apoptosis induced by UV radiation as afunction of its SOD concentration.

[0046]FIG. 3 represents the effects evaluated 72 hours after irradiationof the composition according to the present invention containing 30units per ml of SOD on keratinocytic apoptosis induced by UV radiationas a function of its time of administration.

[0047]FIG. 4 represents the degradation time in gastric medium of thecomposition according to the present invention (B) compared with theuncoated active plant extract (A).

[0048]FIG. 5 represents the degradation time in gastric medium of thecomposition according to the present invention (B) compared with thecomposition according to the present invention homogeneously mixed withfibers (C) or yogurt (D).

[0049]FIG. 6 represents the effect in vivo evaluated 48 hours afterirradiation of the composition according to the present invention (B)taken orally by mice on inflammation induced by UV radiation comparedwith the uncoated active plant extract (A) and with a control (E).

[0050]FIG. 7 represents the effect in vivo evaluated 48 hours afterirradiation of the composition according to the present invention (B)taken orally on inflammation induced by UV radiation compared with thecomposition according to the present invention homogeneously mixed withfibers (C) or yogurt (D) and with a control (E).

[0051]FIG. 8 represents the effect of the semisolid preparation forlocal application containing a composition according to the presentinvention on inflammation induced by UV radiation according to the doseof cream applied and compared with a control.

[0052] The following examples are given as a guide, without limitation.

EXAMPLE 1

[0053] Preparation of a Composition According to the Present Invention

[0054] Preparation of an Active Plant Extract (Described in patentapplication FR2 716 884)

[0055] 5 g of pulp from a hybrid cucumis melo derived from the cell line95LS444 (described in international application WO 92/02622) are groundin a mortar in the cold state. A volume of 50 mM phosphate buffer (pH:7.5; 5EDTA 1 mM; glycerol 5%) equivalent to 3 times the plant mass isadded. After homogenization, the suspension is centrifuged at 5000 g at4° C. for 30 minutes. The supernatant is then recovered and filtered.This crude extract serves for the determination of the superoxidedismutase activity. It is measured with the aid of an SOD 525 assay kitprovided by the company Bioxytech. This method gives results comparableto those obtained by the McCord and Fridovitch method (inhibition by SODof the reduction of cytochrome C by xanthine-xanthine oxidase).

[0056] Preparation of the Composition According to the Present Invention

[0057] The active plant extract prepared above in the form of afreeze-dried powder which is also sold by the company BIONOV under thetrademark EXTRAMEL® is used. This extract, after coating, has an SODactivity of 14 units per mg of extract and also contains catalase andcoenzyme Q10.

[0058] The fat-soluble agent used is hydrogenated palm oil.

[0059] The melting of this agent is carried out in the container (1)(FIG. 1) by heating. The liquid agent thus obtained is deposited byspraying with the aid of a bifluid Schlick nozzle (2) onto the powder ofthe active plant extract suspended in a fluidized bed (7) at lowtemperature (“skin” temperature less than 40° C.) and a low moisturecontent. The low melting point of this coating agent (58 to 61° C.)allows instant solidification of the material without burning the activeplant extract. According to FIG. 1, a ventilator (3), a heater (4), adesaturation device (5) and a filter (6) are used.

[0060] The composition thus obtained contains 20% by weight of theactive plant extract and 80% by weight of the fat-soluble agent.

EXAMPLE 2

[0061] Production of a Semisolid Preparation for Local ApplicationComprising a Composition According to the Present Invention

[0062] The preparation (oil-in-water emulsion) is produced in a mannerwell known to persons skilled in the art using the ingredients and theproportions indicated in Table 1. TABLE 1 Ingredients and proportionsfor the semisolid preparation INGREDIENTS % by weight Refined macadamiaoil (Soetenaey  33% Laboratoire, Fécamp) Kosteran - S/1 G (Verfilco -3.8% Fontenay S/Bois) Kotilen - S/1 (Verfilco-Fontenay 1.2% S/Bois)Water QS 100% Composition according to Example 1 2.2% Preservative(Sepicide; Seppic; 0.3% Castres)

[0063] A semisolid preparation for local application is obtained whichhas the characteristics indicated in the following Table 2. TABLE 2Characteristics of the semisolid preparation for local applicationaccording to Example 2 Appearance Fine emulsion pH 6.93 Stability Stableat room temperature Stable at 40° C. Stable in heat cycle

EXAMPLE 3

[0064] Evaluation of the Protective Activity of the CompositionAccording to Example 1 in Relation to UV Radiation

[0065] In vitro on Normal Human Keratinocytes

[0066] The object of this study is to evaluate the protective effect ofthe composition according to Example 1 in relation to UV radiation whichhas as principle consequences in the epidermis the induction ofpremature death of the keratinocytes by apoptosis (Sun Burn Cell) aswell as a release of soluble mediators which contribute to thedevelopment of a local inflammation.

[0067] Among these proinflammatory mediators whose production appears tobe stimulated by UV radiation, even at very low doses, there may bementioned IL-1α, TNF-α, IL-6 and the free radical NO. The latter is infact produced by the oxidative conversion of arginine to citrulline inthe presence of oxygen via inducible NO-Synthase which is expressedfollowing UV stress. A major proinflammatory mediator, NO appears to bethe main agent responsible for tissue degradations linked to theinflammatory state.

[0068] The protective effect of the active plant extract sold by thecompany BIONOV under the trademark EXTRAMEL® and of the compositionaccording to Example 1 was evaluated in an experimental approach basedon an in vitro model which makes it possible to determine cellularapoptosis as well as the production of proinflammatory mediatorsfollowing UV irradiation.

[0069] In this study, it was chosen to study, in a first instance, theproduction of the proinflammatory cytokines TNF-α and IL-6 and of the NOradical by keratinocytes subjected to UVB radiation.

[0070] a) Material and Methods

[0071] Cells

[0072] The keratinocytes are obtained from primary cultures of foreskinsand are allowed to proliferate in a conditioned medium (KeratinocyteGrowth Medium (KGM)) for two weeks before use. At confluence, the purekeratinocytes are cultured in 24-well plates at 10⁶ cells/ml in a mediumfree of growth factors in the presence or in the absence of the productto be tested for 24 h and are then stimulated by UV radiation.

[0073] Medium for Evaluating the Effects of the Product

[0074] During the evaluation of the product, the cells are cultured inIscove medium supplemented with antibiotics (100 SOD units (U)/ml ofpenicillin and 100 μg/ml of streptomycin) and fetal calf serum (5%), andthey are brought into contact with the composition according to Example1 at various concentrations (0.1 to 1000 μg/ml) and then stimulated byUV radiation.

[0075] UV Stimulation:

[0076] The cells were irradiated with ″¼ m Oriel Monochromator, 1 KWmercury-xenon arc lamp with a pass band of 5 nm and focused through aluminous liquid guide (5 nm×1 m). The irradiation is carried out at 14.4Kj/m² at 310 nm for a period of 10 min, this type of irradiationcorresponding to a UVB-type irradiation.

[0077] The determination of the production of the proinflammatorymediators TNF-α, IL-6 and NO is carried out 48 h after irradiation.

[0078] The percentage of apoptotic cells is evaluated 72 h afterirradiation.

[0079] Measurement of Apoptosis

[0080] At the end of the culture, death by apoptosis is evaluated usingthe Apoptag® kit, the DNA breaks being visualized by fluorescenceemission.

[0081] The number of cells undergoing apoptosis is evaluated by countingthe number of fluorescent cells out of a total of 200 cells.

[0082] Assay of the Inflammation Mediators IL6, TN-α and NO

[0083] The assays of the cytokines TNF-α and IL-6 are carried out onculture supernatants using ELISA assay kits, the detection thresholdsbeing each time less than 10 pg/ml. As regards the production of NO, itis in fact the stable derivatives, i.e. the nitrites, which are measuredby a calorimetric technique using the Griess reagent.

[0084] b) Results

[0085] Apoptosis

[0086] The keratinocytic apoptosis appears to be greatly increased 72 hafter UV irradiation as indicated in the following Table 3. TABLE 3Keratinocytic apoptosis induced by UV irradiation % apoptosis relativeto the total number of cells in Treatment culture Keratinocytes(control) 5 ± 2 Keratinocytes/UV 78 ± 4 

[0087] These results therefore make it possible to institute this modelas a model for evaluating products having a “protective” activityprofile.

[0088] Influence of the concentration of the composition according toExample 1 on the antiapoptotic power

[0089] The composition according to Example 1, tested in a concentrationrange of between 1 and 30 SOD units, is added 24 h before irradiation.Each experiment is carried out in triplicate.

[0090] These effects are evaluated 72 h after irradiation and the % ofcells undergoing apoptosis is evaluated by fluorescence microscopy afterusing the Apoptag® kit (FIG. 2).

[0091] These experiments clearly demonstrate that the compositionaccording to the invention has a protective activity against thephenomenon of induction of apoptosis post-UV irradiation, when it isadded preventively, that is to say before the irradiation phenomenon.During these experiments, the use of the active plant extract Extramel®at a dose of 30 SOD units produces an effect equivalent to 0.3 SOD unitsof the composition according to Example 1 which suggests that the activeplant extract, when it is delivered intracellularly by the use of theprotection in the form of a coating with the aid of a fat-soluble agentaccording to the present invention, is 100 times more effective than thefree active plant extract.

[0092] Influence of the conditions for adding the composition accordingto Example 1 to the culture medium

[0093] The concentration of the composition according to Example 1selected for this study is 30 SOD units/ml (FIG. 3).

[0094] To determine the capacity of this composition to act on apoptosisinduced by UV, preventively, immediately and post-irradiation, it wasadded 24 h before irradiation, at the beginning of irradiation andfinally 24 h after irradiation, respectively.

[0095] Under the conditions of the study, a marked reduction inapoptosis is observed when the composition is added 24 h beforeirradiation (T−24 H).

[0096] Added at the beginning of irradiation (T0), the antiapoptoticeffect of the composition according to Example 1 appears to be reducedand this effect is even more reduced when this composition is added 24 hafter irradiation (T+24 H).

[0097] The product therefore appears to be very effective preventively.

[0098] Production of Cytokines and of NO

[0099] The stimulation of human keratinocytes by UV radiation (Table 4)under the conditions used for these experiments induces the productionof the cytokines TNF-α and IL-6 and the production of NO, via theactivation of inducible NO-Synthase in these cells.

[0100] Influence of the conditions for adding the composition accordingto Example 1 to the culture medium on the production of theproinflammatory mediators

[0101] The capacity of the composition according to the presentinvention to modulate the production of proinflammatory mediators wasevaluated according to the following experimental conditions:introduction of the composition according to Example 1 24 h beforeirradiation or at the beginning of irradiation or 24 h afterirradiation.

[0102] The composition according to Example 1 is tested at 30 SODunits/ml. The assay of the mediators was carried out in triplicate. Theresults are presented in Table 4. TABLE 4 Assay of the proinflammatorymediators 48 h after UV irradiation Treatment TNF (pg/ml) IL-6 (pg/ml)NO₂ ⁻ (μM) None or the Not detectable Not detectable 0.65 ± 0.25composition of in the 3 in the 3 Example 1 alone experiments experimentsUV 310 ± 75 255 ± 30 10 ± 2  UV + composition  95 ± 12 100 ± 18 1.5 ±2   according to Example 1 administered at T − 24 h UV + composition 125± 6  108 ± 4  6.5 ± 2   according to Example 1 administered at T0 UV +composition 306 ± 17 212 ±  8 9.2 ± 2   according to Example 1administered at T + 24 h

[0103] A reduction in the production of the proinflammatory cytokinesand in the nitro derivatives is observed when the composition accordingto Example 1 (30 U/ml) is added preventively (T−24 h) whereas thiseffect is reduced when the addition is made immediately (T0) and haslittle effect when the product is added 24 h after UV stimulation (T+24h).

[0104] c) Conclusion

[0105] The production of the proinflammatory mediators as well as thedeath of the cells by apoptosis are phenomena linked to a massiveproduction of free oxygen radicals (ROS) derived from an interactionbetween UV radiation and oxygen O₂. These free radicals are responsiblefor the oxidative stress generated by UV radiation.

[0106] When the physiological antioxidant pool is no longer able tocontrol them, phenomena of cell degeneration accompanied by aninflammatory reaction develop.

[0107] The composition according to the present invention added beforethe UV stress inhibits the expression of the proinflammatory mediatorsand simultaneously reduces the entry of the cells into apoptosis.

[0108] In this study, it therefore clearly appears that the compositionaccording to the present invention exerts a protective effect on thecellular metabolism.

[0109] Indeed, in the presence of the composition according to thepresent invention, the keratinocytic apoptosis induced by UV irradiationappears to be significantly reduced. Likewise, the increase in theproduction of proinflammatory mediators induced by UV irradiationappears to be greatly attenuated (TNF-α, IL-6 and NO).

[0110] Consequently, the composition according to the present inventionprotects the skin tissue from the degeneration phenomena generated byoxidative stress, regardless of the origin thereof, whether physical (UVradiation) or chemical (environmental pollutants such as ozone, carbonmonoxide or heavy metals, and the like), in particular when it isadministered preventively.

[0111] In vivo on Transplants of Human Skin onto “Nude” Mice

[0112] The object of this study is to evaluate the effect, by the oralroute, of an active plant extract alone (Extramel®) or coated with acomposition according to the present invention containing SOD againstinflammation induced in the skin by UV radiation whose main consequencein the epidermis is to induce a release of soluble mediators whichcontribute to the development of a local inflammation.

[0113] Among these proinflammatory mediators, whose production appearsto be stimulated by UV radiation, even at very low doses, there may bementioned TNF-α and the free radical NO. The latter is in fact producedby the oxidative conversion of arginine to citrulline in the presence ofoxygen via inducible NO-Synthase which is expressed following UV stress.A major proinflammatory mediator, NO appears to be the main agentresponsible for tissue degradations linked to the inflammatory state.

[0114] The protective effect of the composition according to Example 1(mixed or otherwise with food fibers and/or yogurt) was evaluated in anexperimental approach based on the following model: in vivo on explantsof normal human skin which are transplanted onto nude mice andirradiated with UVB, only the portion of human skin being taken intoaccount in the irradiation and in the evaluation of efficacy produced.

[0115] a) Materials and Methods

[0116] UV Stimulation

[0117] Mice carrying human skin were irradiated with ″¼ m OrielMonochromator, 1 KW mercury-xenon arc lamp with a pass band of 5 nm andfocused through a luminous liquid guide (5 nm×1 m). The irradiation iscarried out at 14.4 Kj/m² at 310 nm for a period of 10 min, this type ofirradiation corresponding to a UVB-type irradiation.

[0118] The determination of the production of the proinflammatorymediators TNF-α and NO is carried out 48 h after irradiation on the skinexplants.

[0119] Assay of the Inflammation Mediators TNF-α and NO

[0120] The assay of the cytokines TNF-α is carried out on culturesupernatants using ELISA assay kits, the detection thresholds being eachtime less than 10 pg/ml. As regards the production of NO, it is in factthe stable derivatives, i.e. the nitrites, which are measured by acolorimetric technique using the Griess reagent.

[0121] b) Preliminary Results in Vivo

[0122] A comparison was made (FIG. 4) between the degradation of theactive plant extract marketed by the company BIONOV under the trademarkEXTRAMEL® (A) and the composition according to Example 1 (B) (that is tosay the same active plant extract, but this time protected by means ofits coating with a fat-soluble agent). If the active plant extract isdegraded in the minutes following its introduction into a mediummimicking the gastric juice (pH 1, trypsin and pepstatin), thecomposition according to Example 1 has an increased resistance. Indeed,this composition releases SOD progressively between 5 and 30 min (FIG.4). However, while this composition does not exhibit absolute protectionwith respect to the digestive process, it appears nevertheless that itcan be used as a food additive, in particular in fibers (C) and/oryogurts (D), because in this case the protection of the SOD activity isoptimized (FIG. 5).

[0123] These results demonstrate that the SOD activity contained in theactive plant extract may be taken by the oral route in compositionsaccording to the present invention which may, in addition, be mixed withsome food products (such as yogurt and/or fibers) and thus protect theSOD activity. This oral delivery mode should therefore make it possibleto demonstrate the functional activity of this SOD activity in thefunctional food.

[0124] The type of treatment followed is the following: the animals aretreated with 10 SOD units/mouse each day for 2 weeks before irradiationwith UV radiation.

[0125] In a model of human skin transplant onto “nude” mice, it isdemonstrated that the oral taking of the composition according to thepresent invention protects against skin impairments induced by UVradiation. Thus, UV irradiation of human skin transplanted onto “nude”mice induces the production of inflammatory mediators such as TNF-α. Theoral taking of the free active plant extract (EXTRAMEL®: A) has noeffect whereas the taking of the composition according to Example 1 (B)has a small effect (FIG. 6). When the composition according to Example 1is homogeneously mixed with dietary fibers (C) and/or yogurts (D), agreater protective effect may also be demonstrated (FIG. 7): 35%inhibition for the fibers/composition product according to Example 1 (C)and 25% for the yogurts/composition product according to Example 1 (D)compared with a control (E).

[0126] These preliminary results demonstrate that the taking of thecomposition according to the present invention complexed with other foodproducts (fibers and yogurts) produces a protective effect in vivoagainst the development of a skin inflammation induced by UV radiation.These results strengthen the functional food concept by developingdietary principles which protect the dietary active agents fromdegradation: thus the composition according to the present invention hasa maximum protective activity (although such an activity also exists forthe composition according to the present invention alone by the oralroute) when it is combined with other food products and thereforepositions itself as a functional food additive.

Example 3

[0127] Evaluation of the Protective Activity of the Free Active PlantExtract or in the Composition According to the Present Invention inRelation to Inflammation Associated with the Allergy Phenomenon

[0128] The object of this study is to evaluate the protective effect ofthe active plant extract alone or protected in the composition accordingto the present invention, in relation to inflammation induced byallergenic substances, chemical compounds of any nature.

[0129] The inflammatory response which results from the synergy betweenmultiple biochemical mechanisms is triggered by the release, at thelevel of the site attacked, of numerous proinflammatory mediators (TNF-αNO, IL-1, IL-8, IL-6, and the like) some of which, like NO, contributetoward increasing cell death by apoptosis. The latter, a majorproinflammatory mediator, appears moreover to be the main agentresponsible for tissue degradations linked to the inflammatory state.

[0130] Accordingly, to evaluate the protective effect of this extractagainst allergic stress, we chose an experimental approach based on anin vitro model which makes it possible to determine cellular apoptosisas well as the production of proinflammatory mediators.

[0131] In this study, we chose to study, in a first instance, theproduction of the proinflammatory cytokines TNF-α and IL-6 and theproduction of the NO radical.

[0132] Materials and Methods

[0133] a) Products Studied

[0134] The products used are: the composition according to Example 1 andan active plant extract of Cucumis Melo obtained from the company BIONOV(EXTRAMEL®). This is therefore the free extract and the extractprotected by coating with a fat-soluble agent according to the presentinvention.

[0135] b) Course of the Study

[0136] Cells

[0137] The keratinocytes are obtained from primary cultures of foreskinsand are allowed to proliferate in a conditioned medium (KGM medium) fortwo weeks before use. At confluence, the pure keratinocytes are culturedin 24-well plates at 10⁸ cells/ml in a growth medium in the presence orin the absence of the product to be tested for 24 h, and are thenstimulated.

[0138] Medium for Evaluating the Effects of the Products Studied

[0139] During the evaluation of the product, the cells are cultured inIscove medium supplemented with antibiotics (100 SOD units/ml ofpenicillin and 100 μg/ml of streptomycin) and fetal calf serum (5%), andthey are brought into contact with the products studied at variousconcentrations (0.01 to 100 μg/ml) and then stimulated under theconditions presented below.

[0140] “Allergic” Stimulation

[0141] To mimic the development of an inflammatory reaction of allergicorigin, the cells are preactivated with IL-4 (10 ng/ml) for 48 h so asto induce CD23 (receptor with low affinity for IgE) and then challengedstimulated with the IgE (IgE/anti-IgE) immune complexes. The cells thusactivated are then maintained in culture for 48 h before collecting thesupernatants in order to assay the mediators TNF-α, IL-6 and NO. Thepercentage of apoptotic cells is evaluated 72 h after stimulation.

[0142] Measurement of Apoptosis

[0143] At the end of the culture, death by apoptosis is evaluated usingthe Apoptag® kit, the DNA breaks being visualized by fluorescenceemission. The number of cells undergoing apoptosis is thereforeevaluated by counting the number of fluorescent cells out of a total of200 cells.

[0144] Assay of the Inflammation Mediators IL-6, TNF-α and NO

[0145] The assays of the cytokines TNF-α and IL-6 are carried out onculture supernatants using ELISA assay kits, the detection thresholdsbeing each time less than 10 pg/ml. As regards the production of NO, itis in fact the stable derivatives, i.e. the nitrites, which are measuredby a colorimetric technique using the Griess reagent.

[0146] Results

[0147] a) Apoptosis

[0148] 72 h after activation of the cells with the IgE/anti-IgE complex,significant apoptosis is observed. The results are assembled in Table 5.TABLE 5 Percentage of apoptotic cells after simulation of aninflammation of allergic origin % apoptosis relative to the total numberof cells Treatment cultured Nontreated keratinocytes 3 ± 2Keratinocytes-CD23 (treated 8 ± 1 with IL-4) Keratinocytes-CD23 + IgE IC45 ± 4 

[0149] These results demonstrate that the induction of an inflammatoryreaction of allergic origin induces a significant increase in death byapoptosis of the stimulated keratinocytes. This therefore makes itpossible to institute this model as a model for the evaluation ofproduct having a “protective” activity profile.

[0150] b) Antiapoptotic Effect of the Active Plant Extract AloneExtramel(: Influence of the Addition Conditions

[0151] To determine the capacity of the active plant extract aloneEXTRAMEL® to modulate apoptosis induced by an allergic stress,preventively, immediately or following stress, this extract was added 24h before stimulation, at the time of stimulation and finally 24 h after,respectively.

[0152] The active plant extract alone EXTRAMEL® is introduced at 10μg/ml. This experiment was carried out in triplicate. The results areassembled in Table 6. TABLE 6 Percentage of apoptotic cells in the“allergic inflammation” model Experiment Experiment Experiment IgE IC 12 3 Mean Without active 30 25 38 31 plant extract EXTRAMEL ® Activeplant 11 15 21 15.6 extract alone EXTRAMEL ® (T − 24 h) Active plant 3220 26 26 extract alone EXTRAMEL ® (T0) Active plant 33 27 35 31.6extract alone EXTRAMEL ® (T + 24 h)

[0153] Under the conditions of the experiment, a marked reduction inapoptosis is observed when the active plant extract alone EXTRAMEL® isadded 24 h before irradiation. The active plant extract alone EXTRAMEL®therefore exerts a protective activity.

[0154] c) Production of Cytokines and of NO

[0155] The capacity of the active plant extract alone EXTRAMEL® tomodulate the production of proinflammatory mediators by sensitizedkeratinocytes was evaluated according to the following experimentalconditions: introduction of the active plant extract alone EXTRAMEL® 24h before stimulation, or just before stimulation, or 24 h afterirradiation.

[0156] The active plant extract alone EXTRAMEL® is introduced at 10μg/ml. Each experiment was carried out in triplicate. The results areassembled in Table 7. TABLE 7 Assay of the mediators 48 h after allergicstimulation Treatment TNF (pg/ml) IL-6 (pg/ml) NO₂ ⁻ (μM) None or activeNot Not Mean: 1.00 plant extract detectable detectable alone EXTRAMEL ®in the 3 in the 3 experiments experiments IgE IC Mean: 632 Mean: 428Mean: 17.3 IgE IC Mean: 152 Mean: 208 Mean: 3.6  Active plant extractalone EXTRAMEL ® (T − 24 h) IgE IC Mean: 620 Mean: 525 Mean: 9   Activeplant extract alone EXTRAMEL ® (T0) IgE IC Mean: 655 Mean: 502 Mean:10.1 Active plant extract alone EXTRAMEL ® (T + 24 h)

[0157] The allergic stress, under the conditions used for theseexperiments, stimulates the production of the cytokines TNF-α and IL-6and the production of NO, via the activation of inducible NO-Synthase inthese cells.

[0158] Moreover, a reduction in the production of the proinflammatorycytokines and of the nitro derivatives is observed when the active plantextract alone EXTRAMEL® (10 μg/ml) is added preventively (T−24 h)whereas the addition immediately (T0) or 24 h after the stimulations(T+24 h) has much less effect on the production of these mediators.

[0159] d) Comparison of the Effect of the Active Plant Extract AloneExtramel® with the Composition According to Example 1

[0160] The composition according to Example 1 was tested under theexperimental conditions described above and, in the same quantity,exhibits better efficacy than the active plant extracts alone EXTRAMEL®.The results are assembled in Table 8. TABLE 8 Comparison of the effectof the active plant extract alone EXTRAMEL ® and the compositionaccording to Example 1 Toxicity Treatment % mortality TNF (pg/ml) NO₂ ⁻(μM) None Active plant 5 Not detectable Mean: 0.8  extract alone 5 Notdetectable Mean: 1.00 EXTRAMEL ® Composition 5 Mean: 50  Mean: 1.0 according to Example 1 IgE IC 35 Mean: 508 Mean: 19.3 IgE IC 20 Mean:168 Mean: 5.6  Active plant extract alone EXTRAMEL ® IgE IC 10 Mean: 62 Mean: 0.8  Composition according to Example 1

[0161] Conclusion

[0162] It appears clearly in this study that the active plant extractalone EXTRAMEL® exerts a protective effect against the inflammatoryphenomenon linked to an allergic response and that the compositionsaccording to the invention, that is to say this extract protected by acoating with the aid of a fat-soluble agent according to the invention,are more effective than the active plant extract alone.

[0163] The composition according to the invention significantly reducescellular apoptosis induced by allergic stress.

[0164] This effect appears to be linked to the regulation of theinflammatory phenomenon linked to the allergy since the compositionaccording to the invention significantly reduces the production ofTNF-α, of IL-6 and of NO by the sensitized keratinocytes.

[0165] An active agent capable of reducing inflammation linked to theallergy phenomenon, the composition according to the invention protectsthe skin tissue from inflammation caused by allergenic compounds,chemical substances of any nature (metals such as nickel, proteinsubstances, cosmetic excipients, and the like) present in products fordaily use as well as in environmental pollutants.

EXAMPLE 4

[0166] Evaluation of the Protective Activity of the Cream According toExample 2 Against UV Radiation

[0167] The evaluation model is performed on a normal skin explantirradiated or otherwise with UV radiation (A+B).

[0168] The production of TNF was chosen as inflammation marker.

[0169] The product tested is the semisolid preparation for localapplication according to Example 2, that is to say containing acomposition according to Example 1.

[0170] The doses tested on the skin before exposure to UV radiation are:

[0171] SOD1: 1 mg/cm² of skin,

[0172] SOD2: 0.5 mg/cm² of skin,

[0173] SOD3: 0.1 mg/cm² of skin and

[0174] SOD4: 0.05 mg/cm² of skin.

[0175] The TNFs are measured after exposure to UV radiation with the aidof an ELISA assay kit.

[0176] The results (FIG. 8) demonstrate a protective skin activity ofthe composition according to the present invention formulated fortopical application against inflammation induced by UV irradiation, inparticular at doses greater than 0.05 mg/cm² of skin.

1. Composition comprising an active plant extract containing superoxidedismutase, said extract being coated and/or microencapsulated in afat-soluble agent based on a fatty substance.
 2. Composition accordingto claim 1, characterized in that the plant extract is a protein extractof cucumis melo, in particular in powdered form.
 3. Compositionaccording to claim 2, characterized in that the cucumis melo is aprogeny of the cell line 95LS444 or of one of the hybrid lines derivedfrom 95LS444.
 4. Composition according to any one of the precedingclaims, characterized in that the fat-soluble agent has a melting pointof between about 40 and about 80° C., advantageously of between about 55and about 60° C.
 5. Composition according to any one of the precedingclaims, characterized in that the fat-soluble agent is chosen from thegroup consisting of hydrogenated oils; palm oil or oil from the heart ofpalm fruit; hydrogenated seeds; stearates, in particular chosen fromstearins, stearic acid and its derivatives; waxes; fatty acidmonodiglycerides; saturated C₁₄-C₂₀ fatty acid triglycerides andmixtures thereof.
 6. Composition according to claim 5, characterized inthat the fat-soluble agent is hydrogenated oil based on plant oil, inparticular chosen from the group consisting of hydrogenated coconut oil,hydrogenated palm oil, hydrogenated soybean oil or hydrogenated rapeseedoil.
 7. Composition according to claim 6, characterized in that thefat-soluble agent is hydrogenated palm oil.
 8. Composition according toany one of the preceding claims, characterized in that the plant extractis present at a concentration of between 1 and 90% by weight relative tothe total weight of the composition, advantageously between 20 and 50%by weight relative to the total weight of the composition.
 9. Method formanufacturing a composition according to claims 1 to 8, characterized inthat it comprises the following steps: a) melting the fat-soluble agent,b) depositing the liquid obtained in step a) on the active plant extractcontaining superoxide dismutase.
 10. Method according to claim 9,characterized in that the plant extract is present in the form of apowder and that step b) is carried out in a fluidized bed,advantageously at low temperature and a low moisture content.
 11. Methodaccording to claim 10, characterized in that the temperature of step b)is less than the melting point of the fat-soluble agent and isadvantageously about 40° C.
 12. Pharmaceutical or cosmetic compositionfor topical use comprising a composition according to claims 1 to 8 andan excipient appropriate for topical use for human or animaladministration.
 13. Pharmaceutical, food or cosmetic composition fororal use comprising a composition according to claims 1 to 8 and anexcipient appropriate for oral use for human or animal administration.14. Composition according to claim 13, characterized in that itcomprises, in addition, yogurt and/or fibers, advantageously so as toform a homogenous mixture with the composition according to claims 1 to8.
 15. Composition according to claims 1 to 8, 12 to 14 as a medicament.16. Composition according to claims 1 to 8, 12 to 14 as a medicamentintended for treating or for preventing allergies or for protecting theepidermis from UV radiation.
 17. Use of a composition according toclaims 1 to 8 and 12 for the manufacture of a medicament for topical useintended for treating allergies, eczema, vitiligo, psoriasis, lupus,skin fibrosis, for eliminating melanomas, for improving cicatrizationsof transplants, for preventing allergies or for protecting the epidermisfrom UV radiation.
 18. Use of a composition according to claims 1 to 8,12 to 14 for the manufacture of a medicament intended for protecting theepidermis from UV radiation, for treating or preventing allergies, fortreating asthma, anemia, male sterility, inflammatory diseases such asanthropathy or degenerative diseases due to cellular and/or organicdegeneration, in particular chosen from the group consisting of Crohn'sdisease, Parkinson's disease, Alzheimer's disease, colorectal cancers,fibroses of varied origins, degenerations due to an infectious agentsuch as AIDS or hepatitis C or degenerations linked to the use of amedicament.
 19. Use of a food composition according to claims 1 to 8, 13and 14 as an energy source, as a tonic source or as an aid forrecuperation and for regaining fitness.
 20. Composition according toclaims 1 to 8, 12 to 14 as a medicament, in particular for topical use,intended for treating or for preventing allergies, advantageously forprotecting the epidermis against allergies.
 21. Use of a compositionaccording to claims 1 to 8, 12 to 14 for the manufacture of amedicament, in particular for topical use, intended for treating or forpreventing allergies, advantageously for protecting the epidermisagainst allergies.